This invention relates to a fibrinolytic inhibitor and, more particularly, to a plasminogen activator inhibitor fragment derived from human umbilical vein endothelial cells.
The plasminogen activators are a class of serine proteases that convert plasminogen to the fibrinolytically active enzyme plasmin (fibrinolysin). Upon being thus activated, the plasmin can attack the coagulation proteins of the fibrin clot (thrombus) and thereby disintegrate the clot. Inhibitors normally present in the blood with plasminogen generally retard this reaction. Most of the plasminogen activator in human plasma exists in the form of an enzyme-inhibitor complex (EI).
Human plasma contains two plasminogen activators that are immunologically distinct, namely tissue plasminogen activator (t-PA) and urokinase (UK). t-PA has been demonstrated to have higher affinity for fibrin than UK and, therefore, is a preferable agent for degradation of the fibrin clot. The source of the plasma t-PA has been presumed to be the vascular endothelium and it has been shown that endothelial cells are associated with plasminogen activator activity. Fibrinolytic inhibitors also have been found in human endothelial cell extracts. See, for example, Emeis et al., Biochem. Biophys. Res. Commun. 110(2) 392-398 (1983).
Although it has been established that plasminogen activator inhibitors are present in endothelial cells, highly purified and characterized isolates of these inhibitors or inhibitor fragments are not believed to have been reported prior to the present invention. Availability of such a purified inhibitor from endothelial cells would be useful for regulation of the fibrinolytic reaction. For example, when the administered dose of plasminogen activator turns out to be greater than biologically required to disintegrate the clot, the excess plasminogen acting on circulating fibrinogen. In such instances, the plasminogen activator inhibitor could be given after administration of t-PA or UK to reduce the fibrinogenolysis caused by any excess plasminogen activator. The risk of hemorrhage would thereby be reduced.
Plasminogen activator inhibitors have been obtained from various sources. The best characterized of these inhibitors, known as protease nexin, was isolated from cultured human fibroblasts and has an apparent M.sub.r of about 43 kilodaltons (KDa). Scott et al., J. Biol. Chem. 260(11), 7029-7034 (1985). It is distinguished by its acid lability, its ability to inhibit both plasminogen activators and plasmin, its relatively high pI (7.5-7.8) and by the stimulatory effect of heparin on its activity.
Plasminogen activator inhibitor also has been partially purified from bovine aortic endothelial cells and has been reported to have a M.sub.r of about 50,000 (50 KDa). Loskutoff et al., Proc. Nat. Acad. Sci. USA 80., 2956-2960 (1983); van Mourik et al., J. Biol. Chem. 259(23), 14914-14921 (1984); and Hekman et al., Ibid. 260 (21), 11581-11587 (1985).
A placental type inhibitor having a M.sub.r of about 50,000 and a pI of about 4.8-4.9 has been described by Lecander et al., Brit. J. Haematol. 57, 407-412 (1984); and Astedt et al., Thromb. Haemostas. 53(1), 122-125(1985). It inhibits t-PA and UK but not plasmin, and it appears to be immunologically distinct from inhibitors found in endothelial cells. The same inhibitor is produced by monocytes and macrophages. See, for example, Vassalli et al., J. Exp. Med. 159, 1653-1668(1984).
Various reports on plasminogen activator inhibitor in human umbilical endothelial cells have been published by Emeis et al., Biochem. Biophys. Res Commun. 110(2), 392-398(1983); Levin, Proc. Nat. Acad. Sci. USA 80, 6804-6808 (1983); Sprenger et al., Biochim. Biophys. Acta 801, 163-170 (1984); Philips et al., Ibid. 802, 99-110 (1984); and other scientists. The molecule acts on t-PA and UK but not on plasmin. It is acid stable and has a M.sub.r of about 50 KDa prior to binding to protease.
Recently, it was reported that a heterologous antiserum was raised against bovine endothelial plasminogen activator inhibitor and used to screen a human endothelial cDNA expression library in E coli. A cDNA insert of an antigen-producing clone was found to hybridize with a human endothelial mRNA, and a synthetic oligonucleotide derived from the insert was used to detect homologous clones. DNA sequencing revealed that the cloned cDNA's had a striking homology with members of the serine protease inhibitor family, namely antithrombin III, .alpha..sub.2 -antiplasmin and .alpha..sub.1 -antitrypsin. Pannekoek, J. Cell. Biochem. Supp. 10A, Absts. 15th Ann. Meeting, UCLA Symposia on Molecular & Cellular Biology, Jan. 20-Feb. 15, 1986, Abst. E119, p. 277. At the latter meeting it was also reported that the plaminogen activator inhibitor gene from bovine aorta endothelial cells was cloned and sequenced. Loskutoff, Ibid. Abst. E5, p. 231.